Procalcitonin (PCT) is known as a biomarker, that reflects the presence and severity of local and systemic bacterial infections, i.e. sepsis (Assicot et al., Lancet 1993; 341:515-8; Muller et al., Crit Care Med 2000; 28:977-83; Harbarth et al., Am J Respir Crit Care Med 2001; 164:396-402; Becker et al., Crit Care Med 2008; 36:941-52; Becker et al, J Clin Endocrinol Metab 2004; 89:1512-25; Nobre et al., Am J Respir Crit Care Med 2008; 177:498-505; Christ-Crain et al., Lancet 2004; 363:600-7; Stolz et al., Chest 2007; 131:9-19; Christ-Crain et al, Am J Respir Crit Care Med 2006; 174:84-93; Briel et al., Arch Intern Med 2008; 168:2000-7; discussion 7-8).
Antigen-specific antibodies are a key tool for the development of immunoassays. Several antibodies against PCT-derived peptides have been described, which have been used in immunoassays to detect PCT, but only few have been tested for their use in sandwich immunoassays to detect native PCT (Table 1). Sandwich immunoassays employing antibodies against the calcitonin- and katacalcin moieties of PCT have been developed to measure PCT in human samples on a routine basis.
For conditions associated with elevated PCT concentrations (excluding medullary thyroid carcinoma), especially bacterial infections and sepsis, it is believed that not only full-length PCT (ca. 13 kDa), but also PCT-derived fragments are present in the blood circulation of patients. Particularly, proteolytic cleavage just upstream from the calcitonin moiety of PCT has been discussed to occur (Muller, et al. Crit Care Med 2000; 28:977-83; Whang et al., J Clin Endocrinol Metab 1998; 83:3296-301), which would lead to two fragments (both ca. 6-7 kDa). However, experimental evidence on this is sparse: Circulating PCT has been isolated from sepsis patients by affinity chromatography using an antibody directed against the calcitonin moiety of PCT, and it has been concluded that PCT3-116 is the major circulating PCT species (Weglohner et al., Peptides 2001; 22:2099-103.). However, several selection steps were performed in this analysis, i.e. only peptides with a calcitonin-containing epitope were purified, and not all relevant fractions from the subsequent reversed-phase HPLC were analyzed Immunoassays for PCT also have not been suitable to address the question of PCT-fragmentation, because either competitive assays involving a single antibody were used (Whang, et al. J Clin Endocrinol Metab 1998; 83:3296-301), or sandwich immunoassays involving two antibodies with epitopes located closely to each other in the C-terminal half of PCT and not covering a broad moiety of PCT were used (Morgenthaler et al., Clin Chem 2002; 48:788-90).
Antibodies against the very N-terminus of PCT have been used in conjunction with an antibody against the katacalcin moiety of PCT in a sandwich assay to detect in samples of septic patients PCT species with an intact N-terminus (DE 10 2007 009 751). N-terminally intact PCT species were found to have different in vivo kinetics than PCT immunoreactivity which was detected with a sandwich immunoassay employing antibodies against the calcitonin- and katacalcin moieties of PCT. Additionally, these N-terminally intact PCT species were found to make up only ca. 10-20% of PCT immunoreactivity which was detected with a sandwich immunoassay employing antibodies against the calcitonin- and katacalcin moieties of PCT. It is not clear, however, at which site(s) between the very N-terminus of PCT and the calcitonin moiety proteolytic cleavage(s) occur(s), which lead(s) to the different concentrations of analytes observed. While it can be assumed that PCT1-116 is cleaved N-terminally by the action of DPP IV leading to PCT3-116 (Weglohner, et al. Peptides 2001; 22:2099-103; Wrenger et al., FEBS Lett 2000; 466:155-9), it is unclear, whether additionally or alternatively PCT1-116 can be cleaved at another site in the middle of the molecule.
Thus, it is unclear, whether an antibody having an epitope roughly upstream from the calcitonin moiety (precisely: upstream from position 53) of PCT, which does not include the very N-terminus of PCT (i.e. position 1 of PCT1-116), in conjunction with an antibody having another epitope, for example an epitope downstream from position 53 (as for instance an epitope within the calcitonin- or katacalcin moiety of PCT), can be used in a sandwich immunoassay to detect native PCT in a patient sample comparably as a sandwich immunoassay employing antibodies having an epitope within the calcitonin moiety of PCT and an antibody with an epitope downstream of that, as for instance an antibody with an epitope within the katacalcin moiety of PCT. Such sandwich immunoassay has been recently described using recombinant PCT as analyte, but recovery of native PCT from patient samples has not been evaluated, and the potential issue of PCT fragmentation has not even been discussed our speculated about (Kramer et al., Anal Bioanal Chem 2008; 392:727-36).
The present invention is partially based on the surprising finding of the inventors that antibodies directed against epitopes contained in amino acid positions 2-52 of Procalcitonin are suitable for measuring PCT using sandwich immunoassays, since PCT is not cleaved in the middle of the molecule.